Patent application title: MODIFIED POLYPEPTIDE HAVING HOMOSERINE ACETYLTRANSFERASE ACTIVITY AND MICROORGANISM EXPRESSING THE SAME
Inventors:
So Young Kim (Gyeonggi-Do, KR)
So Young Kim (Gyeonggi-Do, KR)
Yong Uk Shin (Gyeonggi-Do, KR)
Yong Uk Shin (Gyeonggi-Do, KR)
Chang Il Seo (Incheon, KR)
Chang Il Seo (Incheon, KR)
In Kyung Heo (Seoul, KR)
In Kyung Heo (Seoul, KR)
Ju Eun Kim (Seoul, KR)
Hyun Ah Kim (Jeollabuk-Do, KR)
Han Jin Lee (Seoul, KR)
Han Jin Lee (Seoul, KR)
Kwang Ho Na (Seoul, KR)
Kwang Ho Na (Seoul, KR)
Sung Kwang Son (Seoul, KR)
Sung Kwang Son (Seoul, KR)
Assignees:
CJ CHEILJEDANG CORPORATION
IPC8 Class: AC12N910FI
USPC Class:
435116
Class name: Micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing alpha or beta amino acid or substituted amino acid or salts thereof alanine; leucine; isoleucine; serine; homoserine
Publication date: 2013-10-17
Patent application number: 20130273615
Abstract:
The present invention relates to a polypeptide that is modified to have
homoserine O-acetyltransferase activity, and in particular, the present
invention provides a modified polypeptide having homoserine
O-acetyltransferase activity, in which the amino acid at position 111 of
a polypeptide having homoserine succinyltransferase activity is
substituted with other amino acid.Claims:
1. A modified polypeptide having homoserine O-acetyltransferase activity
having the amino acid sequence of SEQ ID NO: 17 or at least 95%
homologous thereto, in which the amino acid at position 111 from the
start amino acid methionine, of the sequence is substituted with glutamic
acid.
2. The modified polypeptide according to claim 1, wherein the amino acid at position 112 of the polypeptide is further substituted with threonine or histidine.
3. The modified polypeptide according to claim 1, wherein the modified polypeptide has amino acid sequence of SEQ ID NO: 18.
4. The modified polypeptide according to claim 1, wherein the modified polypeptide exhibits resistance to feedback regulation by methionine, through substitution of amino acids.
5. The modified polypeptide according to claim 4, wherein the amino acid is substituted with proline at position 29, substituted with glycine at position 114, substituted with serine at position 140, or one or more combinations of them.
6. The modified polypeptide according to claim 5, wherein the amino acid at position 112 of the polypeptide is further substituted with threonine or histidine.
7. The modified polypeptide according to claim 5, wherein the modified polypeptide has the amino acid sequence of SEQ ID NO: 21.
8. A polynucleotide encoding the modified polypeptide of claim 1.
9. The polynucleotide according to claim 8, wherein the polynucleotide has any one of the nucleotide sequences of SEQ ID NOs: 24 to 29.
10. A recombinant vector comprising polynucleotide sequences operably linked to the polynucleotide of claim 8.
11. A microorganism comprising the polynucleotide of claim 8.
12. The microorganism according to claim 11, wherein the microorganism is additionally modified to have enhanced acetyl-CoA synthetase activity compared to the endogenous acetyl-CoA synthetase activity or additionally modified to have pantothenate kinase activity resistant to feedback inhibition by CoA accumulation.
13. The microorganism according to claim 11, wherein the copy number of one or more genes selected from the group consisting of phosphoenolpyruvate carboxylase-encoding gene (ppc), aspartate aminotransferase-encoding gene (aspC), and aspartate semialdehyde dehydrogenase encoding-gene (asd) is increased, or the promoter of the gene is replaced by an activity-enhanced promoter or is mutated to have enhanced activity.
14. A microorganism transformed with the recombinant vector of claim 10.
15. The microorganism according to claim 14, wherein the microorganism belongs to the genus Escherichia.
16. The microorganism according to claim 15, wherein the microorganism is E. coli.
17. The microorganism according to claim 16, wherein the microorganism is deposited under accession number of KCCM11145P, KCCM11146P, KCCM11147P, KCCM11228P, KCCM11229P or KCCM11230P.
18. A method for producing O-acetyl homoserine, comprising culturing the microorganism of claim 11; and obtaining O-acetyl homoserine that is produced during cultivation of the microorganism.
19. The modified polypeptide according to claim 2, wherein the modified polypeptide has the amino acid sequence of SEQ ID NO: 19 or 20.
20. The modified polypeptide according to claim 6, wherein the modified polypeptide has the amino acid sequence of SEQ ID NO: 22 or 23.
Description:
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a polypeptide that is modified to have homoserine acetyltransferase activity, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, a microorganism that is transformed with the recombinant vector, and a method for producing O-acetyl homoserine using the microorganism.
[0003] 2. Description of the Related Art
[0004] Methionine is one of the essential amino acids in the body, and has been widely used as an animal feed and food additive, as well as a component of medical aqueous solutions and other raw materials for medicinal products. Methionine acts as a precursor of choline (lecithin) and creatine, and is also used as a raw material for the synthesis of cysteine and taurine. In addition, it functions as a sulfur donor.
[0005] S-adenosyl-methionine is derived from L-methionine and serves as a methyl donor in the body, and it is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) is/are also found to prevent lipid accumulation in the liver and arteries and to be effective for the treatment of depression, inflammation, liver diseases and muscle pain.
[0006] Methionine can be chemically or biologically synthesized to be used in animal feed, food and medicines.
[0007] In the chemical synthesis, L-methionine is mostly produced by hydrolysis of 5-(β-methylmercaptoethyl)hydantoin. However, the chemically synthesized methionine has a disadvantage of only being produced as a mixed form of L-type and D-type.
[0008] With regard to biological synthesis of L-methionine, U.S. Patent Publication No. US2005/0054060A1 describes a method of synthesizing homocysteine or methionine directly using H2S or CH3SH, while not using cysteine, by modifying cystathionine synthase for the preparation of microorganisms. In this method, modified cystathionine synthase is directly introduced into cells to synthesize methionine according to intracellular methionine synthesizing process. However, there are practical problems in that this method produces only a small amount of methionine because of inhibitory actions of synthesized methionine resulting from using intracellular methionine metabolic pathways, and H2S or CH3SH also causes cytotoxicity.
[0009] To solve these problems, the present inventors had developed a two-step process of converting L-methionine precursor into L-methionine by enzyme reaction (PCT/KR2007/003650). This two-step process can solve the above problems of cytotoxicity of H2S or CH3SH and metabolic process inhibition by produced L-methionine. Moreover, this process is characterized in that it is very efficient to produce only L-methionine selectively, and not a mixed form of D-methionine and L-methionine.
[0010] In this two-step process, O-succinyl homoserine and O-acetyl homoserine can be used as the methionine precursor. During conversion reaction of methionine, O-acetyl homoserine is advantageous over O-succinyl homoserine in terms of production yield of precursor to methionine ratio. Specifically, 0.91 mole of methionine can be produced from 1 mole of O-acetyl homoserine whereas only 0.67 mole of methionine can be produced from 1 mole of O-succinyl homoserine. Thus, production cost of the final product methionine can be reduced by using O-acetyl homoserine as the methionine precursor, and high production yield of O-acetyl homoserine is a crucial factor for the mass-production of methionine.
[0011] Meanwhile, use of the O-acetyl homoserine or O-succinyl homoserine as the methionine precursor depends on the type of microorganisms. In detail, microorganisms belonging to the genus Escherichia, Enterobacteria, Salmonella, and Bacillus produce O-succinyl-homoserine from homoserine and succinyl-coA by L-homoserine O-succinyltransferase (Biochemistry. 1999 Oct 26; 38(43): 14416-23), and microorganisms belonging to the genus Corynebacterium, Leptospira, Deinococcus, Pseudomonas, and Mycobacterium produces O-acetyl-homoserine from homoserine and acetyl-coA by L-homoserine O-acetyltransferase (Journal of Bacteriology, March 2002, p. 1277-1286).
[0012] Therefore, expression of O-acetyl homoserine transferase by introduction of metX, a foreign gene, is required for the biosynthesis of O-acetyl homoserine using microorganisms of the genus Escherichia which are used to produce recombinant proteins for experimental and industrial purposes. However, there are problems related to negative attitudes of consumers toward introduction of foreign genes into microorganisms used for the production of food products, and proving safety of introduction of foreign genes.
[0013] Accordingly, the present inventors have made efforts to prepare a strain of the genus Escherichia that produces O-acetyl homoserine advantageous in terms of the production yield without introduction of foreign genes. As a result, they found that homoserine succinyltransferase activity can be converted into homoserine acetyltransferase activity by using a modified polypeptide prepared by substituting glutamic acid for amino acid at position 111 of O-succinyl homoserine transferase which is from E. coli, thereby completing the present invention.
SUMMARY OF THE INVENTION
[0014] An object of the present invention is to provide a modified polypeptide, in which the polypeptide having homoserine O-succinyltransferase activity is converted to have homoserine acetyltransferase activity.
[0015] Another object of the present invention is to provide a polynucleotide encoding the above modified polypeptide.
[0016] Still another object of the present invention is to provide a recombinant vector comprising polynucleotide sequences operably linked to the above polynucleotide.
[0017] Still another object of the present invention is to provide a microorganism comprising the above polynucleotide.
[0018] Still another object of the present invention is to provide a microorganism that is transformed to the recombinant vector operably linked to the above polynucleotide.
[0019] Still another object of the present invention is to provide a method for producing O-acetyl homoserine using the microorganism that expresses the modified polypeptide having homoserine acetyltransferase activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a diagram showing a recombinant vector that is operably linked to a polynucleotide encoding the modified polypeptide according to the present invention;
[0021] FIG. 2 shows homology comparison of the primary amino acid sequences of homoserine O-succinyltransferase between E. coli variants;
[0022] FIGS. 3 and 4 show homology comparisons of the primary amino acid sequences of mutant homoserine O-succinyltransferase resistant to feedback regulation by methionine, in which the primary amino acid sequences of the wild-type homoserine O-succinyltransferase, the feedback regulation-resistant mutant homoserine O-succinyltransferase met10A and met11A disclosed in PCT Publication No. WO 2008/127240, and the feedback regulation-resistant mutant homoserine O-succinyltransferase disclosed in PCT Publication No. WO 2005/108561 were used for comparison; and
[0023] FIG. 5 is a diagram showing the preparation of a FRT-one step deletion cassette by overlapping PCR in order to substitute the pro promoter for the acs promoter in the chromosome.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] In one aspect to achieve the above objects, the present invention provides a modified polypeptide having homoserine O-acetyltransferase activity having the amino acid sequence of SEQ OD No. 17 or at least 95% homologous thereto, in which the amino acid at position 111 from the start point amino acid, methionine, of the sequence is substituted with glutamic acid.
[0025] As used herein, the polypeptide having homoserine O-succinyltransferase activity means a polypeptide having an activity of synthesizing O-succinyl homoserine from homoserine and succinyl-coA present in the methionine biosynthetic pathways, as shown in the following Reaction Scheme.
[0026] Homoserine +Succinyl-CoA ->O-Succinyl-Homoserine
[0027] The polypeptide having homoserine O-succinyltransferase activity may be a recombinant polypeptide which is from a microorganism of the genus Enterobacteria, Salmonella, Pseudomonas, Bacillus, or Escherichia, preferably, a recombinant polypeptide having homoserine succinyltransferase activity which is from a microorganism of the genus Escherichia, and more preferably, a recombinant polypeptide having homoserine O-succinyltransferase activity which is from E. coli.
[0028] In the present invention, the polypeptide having homoserine O-succinyltransferase activity may include a polypeptide having homoserine succinyltransferase activity that is composed of the amino acid sequence of SEQ ID NO: 17 or at least 95% homologous thereto, as long as it has the activity shown in the above Reaction Scheme.
[0029] In Examples of the present invention, the homology of the amino acid sequences of homoserine O-succinyltransferase between different species of E. coli was compared. As a result, there was less than 5% variation in the homoserine O-succinyltransferase polypeptides between different species of E. coli (that is, they have at least 95% homology), but there was no significant difference in the homoserine O-succinyltransferase activity (FIG. 2). These results indicate that the polypeptides having 950 or more homology to the polypeptide of SEQ ID NO: 17 of the present invention also have identical homoserine O-succinyltransferase activity, which is apparent to those skilled in the art and is visualized by the present inventors.
[0030] As used herein, the term "modified polypeptide" means a polypeptide having homoserine O-acetyltransferase activity by substituting a part of the amino acid sequences of the polypeptide having homoserine O-succinyltransferase activity, unlike the wild-type. That is, the modified polypeptide of the present invention means a modified polypeptide having the same activity as in the following Reaction Scheme, which has substrate specificity for acetyl-coA rather than succinyl-coA by substituting a part of the amino acid sequences of the polypeptide having homoserine O-succinyltransferase activity.
Homoserine+Acetyl-CoA→O-AcetylHomoserine
[0031] In the present invention, the above modified polypeptide may be a modified polypeptide in which the amino acid at position 111 of a polypeptide having amino acid sequence of SEQ ID NO: 17 or a polypeptide having 950 or more sequence homology thereto is substituted with glutamic acid (SEQ ID NO.: 18), and the amino acid at position 112 of the polypeptide is further substituted with threonine (SEQ ID NO: 19) or histidine (SEQ ID NO: 20).
[0032] The further substitution of threonine or histidine for the amino acid leucine at position 112 was found to enhance homoserine acetyltransferase activity (Tables 2 and 3).
[0033] According to one preferred embodiment, the above modified polypeptide may be a polypeptide having any one of the amino acid sequences of SEQ ID NOs: 18 to 20.
[0034] In Examples of the present invention, a plasmid capable of expressing a polypeptide wherein the amino acid glycine at position 111 of a homoserine succinyltransferase encoded by metA gene of E. coli composed of the nucleotide sequence represented by SEQ ID NO: 39 is substituted with glutamic acid and a plasmid capable of expressing a polypeptide wherein the amino acid at position 112 in addition to the above substitution, is substituted with threonine or histidine are prepared (Example 2).
[0035] Further, Experimental Examples of the present invention showed that only O-succinyl homoserine was produced by CJM2 pCL_Pcj1_metA(wt) and CJM3 pCL_Pcj1_metA(wt) transformed with a plasmid including the wild type metA gene (SEQ ID NO: 39). In contrast, only O-acetyl homoserine was accumulated by a strain that is transformed with a plasmid including the gene encoding the modified polypeptide of the present invention (Experimental Example 2, Tables 2 and 3).
[0036] Therefore, a microorganism expressing the modified polypeptide of the present invention is advantageous in that it is able to produce O-acetyl homoserine as a methionine precursor capable of high yield production without introduction of foreign genes for homoserine acetyltransferase activity.
[0037] In the present invention, the above modified polypeptide may be resistant to feedback regulation by methionine resulting from substitution of a part of the amino acids of the polypeptide having homoserine succinyltransferase activity. That is, most activity of homoserine succinyltransferase is regulated through feedback inhibition by a small amount of methionine in a medium, and thus the modified polypeptide of the present invention may be resistant to feedback regulation by methionine for the mass-production of O-acetyl homoserine.
[0038] In the present invention, the amino acid substitution to avoid the feedback regulation by methionine may be performed according to the method disclosed in PCT Publication No. WO 2008/127240. In detail, the feedback regulation by methionine may be avoided by substitution of proline for the amino acid at position 29, substitution of glycine for the amino acid at position 114, substitution of serine for the amino acid at position 140 of the polypeptide having homoserine succinyltransferase activity, or one or more combinations of the three amino acid substitutions. Preferably, two or more, and most preferably three amino acids may be substituted.
[0039] According to one preferred embodiment, the modified polypeptide resistant to feedback regulation by methionine may be a modified polypeptide having any one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 21 to 23.
[0040] In Examples of the present invention, the amino acids at position 29, 114 and 140 of the recombinant polypeptide having homoserine succinyltransferase activity that is encoded by metA gene of E. coli were substituted by proline, glycine, and serine, respectively so as to avoid feedback regulation by methionine. In addition, constructed were plasmids including polynucleotides encoding modified polypeptides having homoserine acetyltransferase activity, which are [pCL_Pcj1_metA#11(EL)] prepared by substitution of glutamic acid for the amino acid at position 111, [pCL_Pcj1_metA#11(ET)] prepared by substitution of glutamic acid and threonine for the amino acids at position 111 and 112, and [pCL_Pcj1_metA#11(EH)] prepared by substitution of glutamic acid and histidine for the amino acids at position 111 and 112(Example 3).
[0041] Further, Experimental Examples of the present invention showed that among the strains expressing modified polypeptides resistant to feedback regulation by methionine, CJM2 pCL_Pcj1_metA(#11)EH and CJM3 pCL_Pcj1_metA(#11)EH strains prepared by substitution of glutamic acid and histidine for the amino acids at position 111 and 112 showed high O-acetyl homoserine productivities of 11.1 g/L and 24.8 g/L, respectively, and these accumulations of O-acetyl homoserine are similar to those by introduction of foreign homoserine acetyltransferase gene (Experimental Example 2, Tables 2 and 3).
[0042] In another aspect, the present invention provides a polynucleotide encoding the modified polypeptide, or a recombinant vector comprising polynucleotide sequences operably linked to the polynucleotide.
[0043] In the present invention, the above polynucleotide is a nucleotide polymer composed of nucleotide monomers covalently bonded in a chain, and examples thereof are DNA or RNA strands having a predetermined or longer length, and it is a polynucleotide encoding the above modified polypeptide.
[0044] In the present invention, the above polynucleotide may be a polynucleotide having any one of the nucleotide sequences of SEQ ID NOs: 24 to 29.
[0045] As used herein, the above term "recombinant vector" is a means for expressing the modified polypeptide by introduction of DNA into a host cell in order to prepare a microorganism expressing the modified polypeptide of the present invention, and the known expression vectors such as plasmid vector, a cosmid vector, and a bacteriophage vector may be used. The vector may be easily prepared by those skilled in the art according to any known method using recombinant DNA technology.
[0046] In the present invention, the recombinant vector may be a pACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322, or pMW118 vector, and preferably the pCL1920 vector.
[0047] The term "operably linked" means that an expression regulatory sequence is linked in such a way of regulating the transcription and translation of a polynucleotide sequence encoding the modified polypeptide, and includes maintaining a precise translation frame in such a way that the modified polypeptide encoded by the polynucleotide sequence is produced when the polynucleotide sequence is expressed under the control of regulatory sequences (including a promoter).
[0048] In still another aspect, the present invention provides a microorganism comprising the polynucleotide encoding the above modified polypeptide and a microorganism that is transformed with the recombinant vector operably linked to the polynucleotide encoding the above modified polypeptide.
[0049] As used herein, the term "transformation" means a method that a gene is introduced into a host cell to be expressed in the host cell. The transformed gene, if it is in the state of being expressed in the host cell, may be inserted in the chromosome of the host cell or may exist independent of the chromosome.
[0050] In addition, the gene includes DNA and RNA as a polynucleotide capable of encoding a polypeptide. The gene can be introduced in any type, as long as it can be introduced in the host cell and expressed therein. For example, the gene may be introduced into the host cell in the type of expression cassette which is a polynucleotide construct including whole elements for expressing the gene by itself. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site and a translation termination signal, which are operably linked to the gene. The expression cassette may be in the type of the expression vector capable of self-replication. The above gene may also be introduced into the host cell by itself or in the type of polynucleotide construct so as to be operably linked to the sequence required for expression in the host cell.
[0051] The above microorganism is a prokaryotic or eukaryotic microorganism that is able to express the modified polypeptide by including the polynucleotide encoding the modified polypeptide or by transformation with the recombinant vector operably linked to the polynucleotide encoding the modified polypeptide, and for example, it may be a microorganism belonging to the genus Escherichia, Bacillus, Aerobacter, Serratia, Providencia, Erwinia, Schizosaccharomyces, Enterobacteria, Zygosaccharomyces, Leptospira, Deinococcus, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Streptomyces, Pseudomonas, Brevibacterium or Corynebacterium.
[0052] In the present invention, the the microorganism is expressing the polypeptide having homoserine O-succinyltransferase activity. For example, it may be a microorganism belonging to the genus Bacillus, Escherichia, Enterobacteria, or Salmonella, preferably a microorganism belonging to the genus Escherichia, and more preferably, E. coli.
[0053] In Examples of the present invention, prepared were E. coli CJM2 pCL_Pcj1_metAEL, CJM2 pCL_Pcj1_metAET, and CJM2 pCL_Pcj1_metAEH strains transformed with the recombinant vector comprising the polynucleotide encoding the modified polypeptide of the present invention (Example 2 and Experimental Example 2), and E. coli CJM2 pCL_Pcj1_metA(#11)EL, CJM2 pCL_Pcj1_metA(#11)ET, and CJM2 pCL_Pcj1_metA(#11)EH strains transformed with the recombinant vector including the polynucleotide encoding the modified polypeptide resistant to feedback regulation by methionine and having homoserine O-acetyltransferase activity of the present invention (Example 3 and Experimental Example 2). Among the above strains, the CJM2 pCL_Pcj1_metA(#11)EL, CJM2 pCL_Pcj1_metA(#11)ET, and CJM2 pCL_Pcj1_metA(#11)EH strains were designated as CA05-0546, CA05-0547 and CA05-0548, respectively and deposited in the Korean Culture Center of Microorganism on Dec. 14, 2010, and assigned the accession numbers, KCCM11145P, KCCM11146P and KCCM11147P, respectively.
[0054] The present invention provides the modified polypeptide having homoserine O-acetyltransferase activity, in which a part of the amino acid sequences of the polypeptide having homoserine O-succinyltransferase activity is substituted. Thus, it is advantageous in that when the modified polypeptide of the present invention is expressed in the microorganism expressing the polypeptide having homoserine O-succinyltransferase activity only, the polypeptide having homoserine O-acetyltransferase activity can be expressed without introduction of a foreign gene such as metX encoding homoserine O-acetyltransferase.
[0055] In the present invention, the above microorganism may be a microorganism that is additionally modified to have enhanced acetyl-CoA synthetase activity or additionally modified to have pantothenate kinase activity resistant to feedback inhibition by CoA accumulation, in order to produce a large amount of O-acetyl homoserine.
[0056] In the present invention, acetyl-CoA synthetase and pantothenate kinase which are from various microorganisms, and genes encoding the proteins having these activities are commonly called acs and coaA, respectively.
[0057] In the present invention, the enhancement of acetyl-CoA synthetase activity may be achieved through enhancement of gene expression by modification of nucleotide sequences of the promoter region and the 5'-UTR region of the acs gene encoding acetyl-CoA synthetase, and the activity of the protein can be enhanced by introducing the mutation in the ORF region of the corresponding gene, and the protein expression level can be enhanced by the introduction of the extra copy of the corresponding gene on the chromosome, or by the introduction of the corresponding gene with the self-promoter or enhanced other promoter in the strain.
[0058] More specifically, the acetyl-CoA synthetase activity may be enhanced through substitution of activity-enhanced promoter, induction of promoter mutation for enhancement of the activity, or an increase in the gene copy number, and therefore, the present invention provides a method for improving O-acetyl homoserine productivity, and E. coli prepared by the method. For the substitution of activity- enhanced promoter, pTac, pTrc, pPro, pR, and pL, which are known to have enhanced activity, may be used.
[0059] According to one preferred embodiment, the present invention provides an O-acetyl homoserine-producing strain, in which the acs gene involved in acetyl-CoA biosynthesis is overexpressed by substituting a constitutive expressing promoter, pro promoter, for its promoter. The pro promoter may be a part or the entire of SEQ ID NO: 30.
[0060] The present invention further provides a microorganism that is introduced with a modified pantothenate kinase resistant to feedback inhibition by CoA accumulation in the CoA biosynthetic pathways. More specifically, the amino acid arginine at position 106 in the amino acid sequence of the pantothenate kinase is substituted by alanine (SEQ ID NO: 40) so that it becomes resistant to feedback inhibition by CoA accumulation, leading to improvement of O-acetyl homoserine productivity.
[0061] In the present invention, the above microorganism may be a microorganism, in which the copy number of one or more genes selected from the group consisting of phosphoenolpyruvate carboxylase-encoding gene (ppc), aspartate aminotransferase-encoding gene (aspC), and aspartate semialdehyde dehydrogenase encoding-gene (asd) is increased, or the promoter of the gene is replaced by an activity-enhanced promoter or is mutated to have enhanced activity.
[0062] In the present invention, the series of enzymes have the activities of synthesizing O-acetyl homoserine from phosphoenolpyruvate, as shown in the following Reaction
[0063] Schemes. Therefore, accumulation of O-acetyl homoserine in cells can be induced by enhancing expression of the genes having these activities.
[0064] Phosphoenolpyruvate carboxylase (ppc)
Phosphoenolpyruvate+H2O+CO2⇄Oxaloaxetate+Phosphate
[0065] Aspartate aminotransferase (aspC)
Oxaloacetate+Glutamate⇄Aspartate+a-ketoglutarate
[0066] Aspartate kinase (thrA)
Aspartate+ATP⇄Aspartyl-4-phosphate+ADP
[0067] Aspartate semialdehyde dehydrogenase (asd)
Aspartyl-4-phosphate+NADPH⇄Aspartate-semialdehyde+Phosphate+- NADP+
[0068] Homoserine dehydrogenase (thrA)
Aspartate-semialdehyde+NADPH⇄Homoserine
[0069] In Reaction Schemes, the thrA gene encoding the bifunctional enzyme, aspartate kinase/homoserine dehydrogenase is previously enhanced through relief of feedback inhibition in the CJM2 strain in Experimental Example 2, and the rest three enzymes can be enhanced through an increase in the gene copy number, substitution of promoter of the above gene to activity-enhancing promoter, or induction of promoter mutation for enhancement of the activity.
[0070] As used herein, the term "increase in the copy number" means additional introduction of a desired gene into the chromosome or by introduction of a plasmid having the gene encoding the corresponding enzyme.
[0071] In Examples of the present invention, a CJM2-AP strain was prepared by deletion of the acs promoter of a metA and metB-deleted CJM2 strain and substitution of the pro promoter therefor, and then transformed to have feedback resistant coaA so as to prepare a CJM2-AP/CO strain having increased Acetyl-coA pool, followed by preparation of a CJM3 strain having two copies of three ppc, aspC, and asd genes. Thereafter, pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET)-introduced CJM3 strains were designated as CA05-0578, CA05-0579 and CA05-0580, respectively and deposited in the Korean Culture Center of Microorganism on Dec. 12, 2011, and assigned the accession numbers, KCCM11228P, KCCM11229P and KCCM11230P, respectively (Experimental Example 2).
[0072] In still another aspect, the present invention provides a method for producing O-acetyl homoserine, comprising the steps of culturing the microorganism comprising the polynucleotide encoding the modified polypeptide or the microorganism that is transformed with the recombinant vector operably linked to the polynucleotide encoding the modified polypeptide, and obtaining O-acetyl homoserine that is produced during the above cultivation of the microorganism.
[0073] In the present invention, production of O-acetyl homoserine using the microorganism expressing the modified polypeptide may be performed with a proper medium and conditions known in the art. It is well understood by those skilled in the art that the culture method may be easily adjusted according to the selected strain.
[0074] Examples of the culture method include, but not limited to, batch, continuous and fed-batch culture. The medium used in the cultivation has to meet the culture conditions for a specific strain.
[0075] The medium used in the present invention may include any one carbon source of sucrose, glucose, glycerol, and acetic acid or combinations thereof, and the nitrogen source to be used is exemplified by organic nitrogen sources such as peptone, yeast extract, beef extract, malt extract, corn steep liquor, and bean flour, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate or combinations thereof.
[0076] The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium-containing salts as a phosphate source. The medium may also include a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and proper precursors may be added as well. The medium or the precursors may be added to the culture by batch-type or continuous type. pH of the culture may be adjusted during the cultivation by adding appropriately a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid, and the generation of foams may be inhibited during the cultivation by using an antifoaming agent such as fatty acid polyglycol ester.
[0077] In order to maintain aerobic conditions of the culture, oxygen or oxygen-containing gas may be injected into the culture. In order to maintain anaerobic and microaerobic conditions, no gas may be injected or nitrogen, hydrogen, or carbon dioxide may be injected. The temperature of the culture may be 27° C. to 37° C., and preferably 30° C. to 35° C. The period of cultivation may be continued as long as the desired material is produced, and preferably for 10 to 100 hours.
[0078] Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
EXAMPLE 1
[0079] Construction of plasmid including homoserine O-succinyltransferase and homoserine O-acetyltransferase
[0080] PCR was performed using the chromosome of E. coli W3110 strain (Accession No. ATCC9637) purchased from American Type Culture Collection as a template and primers of SEQ ID NO: 1 and SEQ ID NO: 2 to amplify the metA gene encoding homoserine O-succinyltransferase.
[0081] The primers used in PCR were prepared based on the sequence of E. coli chromosome of NC 000913 registered in NIH Gene Bank, and the primers of SEQ ID NO: 1 and SEQ ID NO: 2 have EcoRV and HindIII restriction sites, respectively.
TABLE-US-00001 <SEQ ID NO: 1> 5' AATTGATATCATGCCGATTCGTGTGCCGG 3' <SEQ ID NO: 2> 5' AATTAAGCTTTTAATCCAGCGTTGGATTCATGTG 3'
[0082] PCR was performed using the chromosome of Deinococcus radiodurans as a template and primers of SEQ ID NO: 3 and SEQ ID NO: 4 to amplify the metX gene encoding homoserine O-acetyltransferase (SEQ ID NO: 44). The primers of SEQ ID NO: 3 and SEQ ID NO: 4 have EcoRV and HindIII restriction sites, respectively.
TABLE-US-00002 <SEQ ID NO: 3> 5' AATTGATATCATGACCGCCGTGCTCGC 3' <SEQ ID NO: 4> 5' AATTAAGCTTTCAACTCCTGAGAAACGCCCC 3'
[0083] PCR was performed under the following conditions: denaturation at 94° C. for 3 minutes, 25 cycles consisting of denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, and polymerization at 72° C. for 5 minutes, and polymerization at 72° C. for 7 minutes.
[0084] The obtained PCR products were cloned into pCL1920 plasmid containing cj1 promoter (KR 2006-0068505) after treatment of restriction enzymes, EcoRV and HindIII, respectively. E. coli DH5α was transformed with the cloned plasmids, and the transformed E. coli DH5α was selected on LB plates containing 50 μg/ml of spectinomycin so as to obtain plasmids. The obtained plasmids were designated as pCL_Pcj1_metA and pCL_Pcj1_metXdr, respectively.
EXAMPLE 2
[0085] Construction of modified polypeptide having homoserine O-acetyltransferase activity
[0086] The amino acid glycine (Gly) at position 111 of O-succinyltransferase was substituted by glutamic acid (Glu) using the pCL_Pcj1_metA plasmid prepared in Example 1 as a template and a site directed mutagenesis kit (Stratagene, USA) (G111E). The sequences of the used primers are as follows:
TABLE-US-00003 <SEQ ID NO: 5> 5' ttgtaactggtgcgccgctggaactggtggggtttaatgatgtc 3' <SEQ ID NO: 6> 5' gacatcattaaaccccaccagttccagcggcgcaccagttacaa 3'
[0087] The constructed plasmid containing the mutant G111E metA gene was designated as pCL_Pcj1_metA(EL).
[0088] In addition, the amino acid glycine (Gly) at position 111 of O-succinyltransferase was substituted by glutamic acid (Glu), and the amino acid leucine at position 112 of O-succinyltransferase was substituted by threonine (L112T) or histidine (L112H). At this time, the sequences of the used primers are as follows:
[0089] Substitution of threonine for leucine
TABLE-US-00004 <SEQ ID NO: 7> 5' tgtaactggtgcgccgctggaaaccgtggggtttaatgatgtcg 3' <SEQ ID NO: 8> 5' cgacatcattaaaccccacggtttccagcggcgcaccagttaca 3'
[0090] Substitution of histidine for leucine
TABLE-US-00005 <SEQ ID NO: 9> 5' tgtaactggtgcgccgctggaacatgtggggtttaatgatgtcg 3' <SEQ ID NO: 10> 5' cgacatcattaaaccccacatgttccagcggcgcaccagttaca 3'
[0091] Among the constructed plasmids, the plasmid containing the metA gene, in which the amino acid glycine at position 111 was substituted by glutamic acid and the amino acid leucine at position 112 was substituted by threonine, was designated as pCL_Pcj1_metA(ET). Also, the plasmid containing the metA gene, in which the amino acid glycine at position 111 was substituted by glutamic acid and the amino acid leucine at position 112 was substituted by histidine, was designated as pCL_Pcj1_metA(EH).
EXAMPLE 3
[0092] Construction of feedback-resistant modified polypeptide having homoserine O-acetyltransferase activity
[0093] The metA gene having a resistance to feedback regulation by methionine (metA #11) was constructed using the pCL_Pcj1_metA plasmid prepared in Example 1 as a template in the same manner as in Example 2. Specifically, according to the method disclosed in PCT Publication No. WO 2008/127240, serine, glutamic acid, and phenylalanine at position 29, 114, and 140 of O-succinyltransferase were substituted by proline (S29P), glycine (E114G), and serine (F140S), respectively. The sequences of the used primers are as follows.
[0094] Substitution of proline for serine
TABLE-US-00006 <SEQ ID NO: 11> 5' ATGACAACTTCTCGTGCGCCTGGTCAGGAAATTCG 3' <SEQ ID NO: 12> 5' CGAATTTCCTGACCAGGCGCACGAGAAGTTGTCAT 3'
[0095] Substitution of glycine for glutamic acid
TABLE-US-00007 <SEQ ID NO: 13> 5' CGCCGCTGGGCCTGGTGGGGTTTAATGATGTCGCT 3' <SEQ ID NO: 14> 5' AGCGACATCATTAAACCCCACCAGGCCCAGCGGCG 3'
[0096] Substitution of serine for phenylalanine
TABLE-US-00008 <SEQ ID NO: 15> 5' CACGTCACCTCGACGCTGAGTGTCTGCTGGGCGGT 3' <SEQ ID NO: 16> 5' ACCGCCCAGCAGACACTCAGCGTCGAGGTGACGTG 3'
[0097] Each of the mutations was sequentially introduced to construct a plasmid containing the metA(#11) gene with the three mutations, which was designated as pCL_Pcj1_metA#11.
[0098] Subsequently, constructed were plasmids for expressing polypeptides having mutations identical to those of the modified polypeptides having homoserine O-acetyltransferase activity of Example 2 using the prepared pCL_Pcj1_metA#11 plasmid as a template.
[0099] Among the constructed plasmids, the plasmid containing the metA #11 gene, in which the amino acid glycine at position 111 was substituted by glutamic acid, was designated as pCL_Pcj1_metA#11(EL), the plasmid containing the metA #11 gene, in which the amino acid glycine at position 111 was substituted by glutamic acid and the amino acid leucine at position 112 was substituted by threonine, was designated as pCL_Pcj1_metA#11(ET), and the plasmid containing the metA #11 gene, in which the amino acid glycine at position 111 was substituted by glutamic acid and the amino acid leucine at position 112 was substituted by histidine, was designated as pCL_Pcj1_metA#11(EH).
EXPERIMENTAL EXAMPLE 1
[0100] Homology comparison between E. coli homoserine succinyltransferase and feedback-resistant E. coli homoserine succinyltransferase
[0101] The primary amino acid sequences [SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43 in order] of homoserine O-succinyltransferase of E. coli 09:H4 (strain HS), E. coli 0139: H28 (strain E24377A), and E. coli 0157:H7 (strain ATCC8739) variants were compared using CLC Main Workbench (CLC bio, Denmark) program.
[0102] As shown in FIG. 2, less than 5% variations were observed in the primary amino acid sequences of homoserine O-succinyltransferase of the E. coli variants (FIG. 2).
[0103] The primary amino acid sequences of the mutant homoserine O-succinyltransferase resistant to feedback regulation by methionine were also compared using the above program. For comparison, the primary amino acid sequences of the wild-type homoserine O-succinyltransferase, the feedback regulation-resistant mutant homoserine O-succinyltransferase met10A and met11A disclosed in PCT Publication No. WO 2008/127240, and the feedback regulation- resistant mutant homoserine O-succinyltransferase disclosed in PCT Publication No. WO 2005/108561 were used.
[0104] As shown in FIGS. 3 and 4, less than 5% variations were observed in the primary amino acid sequences of the mutant homoserine O-succinyltransferase resistant to feedback regulation by methionine (FIGS. 3 and 4).
[0105] These results indicate that the homoserine O-succinyltransferase polypeptides present in E. coli had 950 or higher homology therebetween, and there was no great difference in homoserine succinyltransferase activity even though less than 50 of sequence difference.
EXPERIMENTAL EXAMPLE 2
[0106] Comparison of substrate specificity and activity between modified polypeptides having homoserine acetyltransferase activity
[0107] 2-1: Preparation of test strains
[0108] 2-1-1) Deletion of metA and metB genes
[0109] In order to compare activities of modified polypeptides producing excessive amounts of O-acetyl homoserine, a strain accumulating homoserine and having a deletion of O-acetyl homoserine utilization was prepared. The metA and metB gene-deleted strain was prepared by the methods of Examples 1-1 to 1-4 described in Publication Patent EP2108693A2, based on the threonine-producing strain, FTR2533 (KCCM 10541) described in PCT/KR2005/00344. The strain was designated as CJM2. CJM2 is a strain that accumulates a large amount of homoserine and produces O-acetyl homoserine or O-succinyl homoserine depending on the gene introduced.
[0110] 2-1-2) Substitution of acs promoter
[0111] For the production of excessive amount of O-acetyl homoserine, production of homoserine and acetyl-CoA must be facilitated. First, to facilitate the supply of acetyl-coA, the promoter of acs (acetyl-coA synthetase) gene was replaced by the constitutive pro promoter of SEQ ID NO: 30 so as to induce constitutive overexpression of the desired gene. For substitution of the promoter, modified FRT-one-step PCR was performed (PNAS (2000) vol.97: 6640-6645). In order to prepare a cassette as shown in FIG. 5, a pKD3 (PNAS (2000) vol.97: 6640-6645)-derived chloramphenicol resistance FRT cassette was subjected to PCR using SEQ ID NO: 31 and SEQ ID NO: 33, and the pro promoter region was subjected to PCR using SEQ ID NO: 32 and SEQ ID NO: 34. Two PCR products were subjected to overlapping PCR to prepare a single cassette (acs promoter deleted-pro promoter substituted cassette) (Nucleic Acids Res. 1988 August 11; 16(15): 7351-7367). PCR was performed under the following conditions: 30 cycles consisting of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 1 minute.
TABLE-US-00009 <SEQ ID NO: 31> 5' AGGGGCTTCATCCGAATTGCGCCATTGTTGCAATGGCGGTGCTGGAGCTGCTTCGAAGTTC 3' <SEQ ID NO: 32> 5' GATATTCATATGGACCATGGCTCGAGCATAGCATTTTTATCC 3' <SEQ ID NO: 33> 5' GGATAAAAATGCTATGCTCGAGCCATGGTCCATATGAATATC 3' <SEQ ID NO: 34> 5' CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATATGTACCTTTCTCCTCTTTA 3'
[0112] The resulting PCR product was electrophoresed on a 1.00 agarose gel, and then DNA was purified from a band of approximately 1.2 kbp. The recovered DNA fragment was electroporated into the CJM2 strain previously transformed with a pKD46 vector (PNAS (2000) vol.97: 6640-6645). Before electroporation, the CJM2 strain transformed with pKD46 was cultivated at 30° C. in LB medium containing 100 μg/L of ampicillin and 5 mM of L-arabinose until OD600 reached 0.6. Then, the cultured strain was washed once with sterilized distilled water and twice with 10% glycerol. Electroporation was performed at 2500 V. The recovered strain was streaked on LB plate medium containing 25 μg/L of chloramphenicol, followed by cultivation at 37° C. overnight. Then, a strain exhibiting resistance to chloramphenicol was selected accordingly.
[0113] PCR was performed using the selected strain as a template and the same primers under the same conditions. The deletion of acs promoter and substitution of pro promoter were identified by confirming the 1.2 kb sized gene on 1.0% agarose gel. The strain was then transformed with pCP20 vector (PNAS (2000) vol.97: 6640-6645) and cultured in LB medium. The final acs promoter deleted and pro promoter substituted strain was constructed in which the gene size was reduced to 150 by on 1.0% agarose gel by PCR under the same experimental conditions, and it was confirmed that the chloramphenicol marker gene was deleted. The constructed strain was designated as CJM2-AP.
[0114] 2-1-3) Substitution of feedback resistant coaA
[0115] In order to prepare CJM2-AP strain having feedback resistant coaA, PCR was performed using w3110 gDNA as a template and the primers of SEQ ID NO: 35 and SEQ ID NO: 36 containing the EcoRI restriction site so as to obtain a coaA gene encoding pantothenate kinase. High-fidelity DNA polymerase PfuUltra® (Stratagene) was used as a polymerase, and PCR was performed under the conditions of 30 cycles consisting of denaturation at 96° C. for 30 seconds; annealing at 50° C. for 30 seconds; and polymerization at 72° C. for 2 minutes.
[0116] After treatment of the obtained coaA gene and pSG76C plasmid (Journal of Bacteriology, July 1997, 4426-4428) with the restriction enzyme EcoRI, they were ligated with each other. E. coli DH5α was transformed with the constructed plasmid, and then the transformed E. coli DH5α was selected on LB plate medium containing 25 μg/ml of chloramphenicol so as to obtain pSG-76C-coaA.
TABLE-US-00010 <SEQ ID NO: 35> 5' ATGAGTATAAAAGAGCAAAC 3' <SEQ ID NO: 36> 5' TTATTTGCGTAGTCTGACC 3'
[0117] pSG-76C-coaA (R106A) was constructed using the obtained pSG-76C-coaA and the primers of SEQ ID NO: 37 and SEQ ID NO: 38 by site directed mutagenesis (Stratagene, USA).
TABLE-US-00011 <SEQ ID NO: 37> 5' GGAAAAGTACAACCGCCgccGTATTGCAGGCGCTATT 3' <SEQ ID NO: 38> 5' AATAGCGCCTGCAATACggcGGCGGTTGTACTTTTCC 3'
[0118] The CJM2-AP strain was transformed with the pSG76C-coaA (R106A) plasmid and cultured in LB-Cm (Yeast extract 10 g/L, NaCl 5 g/L, Tryptone 10 g/L, chloramphenicol 25 μg/L) medium to select chloramphenicol-resistant colonies. The selected transformant is a strain in which pSG76c-coaA (R106A) is primarily inserted into the coaA region of the genome.
[0119] The coaA (R106A) gene-inserted strain was transformed with a pASceP vector (Journal of Bacteriology, July 1997, 4426-4428) expressing the restriction enzyme I-SceI that cleaves the I-SceI site present in pSG76c, followed by selection of strains on LB-Ap (Yeast extract 10 g/L, NaCl 5 g/L, Tryptone 10 g/L, Ampicillin 100 μg/L). The coaA gene was amplified from the selected strain using the primers of SEQ ID NO: 35 and SEQ ID NO: 36, and the substitution of coaA (R106) in the amplified gene was confirmed by macrogen sequencing service (Korea) (Nucleic Acids Research, 1999, Vol.27, No.22 4409-4415). The prepared strain was designated as CJM2-AP/CO. The CJM2-AP/CO strain is a strain having increased homoserine and acetyl-coA pool.
[0120] 2-1-4) Increase in copy number of key genes in homoserine biosynthetic pathways
[0121] Even though the CJM2 or CJM2-AP/CO strain is a strain producing an excessive amount of homoserine, the copy numbers of three genes of ppc, aspC, and asd were increased to more improve homoserine productivity. pSG76c-2ppc, pSG76c-2aspC, and pSG76c-2asd plasmids were constructed by the methods described in Examples <1-1> to <1-3> of Publication Patent No. KR2011-0023703, and the plasmids were introduced into the CJM2-AP/CO strain to prepare a strain having two copies of the three genes by the method of Example <1-5>. The prepared strain was designated as CJM3. CJM3 is a strain that accumulates a large amount of homoserine compared to the CJM2 strain, and produces O-acetyl homoserine or O-succinyl homoserine depending on the plasmid introduced.
[0122] 2-2: Experimental methods and Experimental results
[0123] Two strains of CJM2 and CJM3 were prepared as competent cells, and 9 plasmids of pCL_Pcj1_metX, pCL_Pcj1_metA, pCL_Pcj1_metA(EL), pCL_Pcj1_metA(EH), pCL_Pcj1_metA(ET), pCL_Pcj1_metA#11, pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH), and pCL_Pcj1_metA#11(ET) were introduced into the competent cells by electroporation, respectively.
[0124] Among them, the CJM2 strains introduced with pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH), and pCL_Pcj1_metA#11(ET) were designated as CA05-0546, CA05-0547 and CA05-0548, respectively. They were deposited in the Korean Culture Center of Microorganism on Dec. 14, 2010, and assigned the accession numbers, KCCM11145P, KCCM11146P, and KCCM11147P, respectively.
[0125] Further, the CJM3 strains introduced with pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH), and pCL_Pcj1_metA#11(ET) were designated as CA05-0578, CA05-0579, and CA05-0580, respectively. They were deposited in the Korean Culture Center of Microorganism on Dec. 12, 2011, and assigned the accession numbers, KCCM11228P, KCCM11229P, and KCCM11230P, respectively.
[0126] Thereafter, a flask test was performed to compare the types and productivities of methionine precursors that were produced by each of the strains introduced with 9 types of plasmids. In the flask test, after streaking each strain on LB plates and culturing them in a 31° C. incubator for 16 hours, single colonies were inoculated in 3 ml of LB medium, and then cultured in a 200 rpm/31° C. incubator for 16 hours.
[0127] 25 ml of the methionine precursor production medium of Table 1 was put in 250 ml flasks, and each 500 it of the culture broths was added thereto. Then, the flasks were incubated in a 200 rpm/31° C. incubator for 40 hours, and the type and productivity of methionine precursor produced by each of the plasmid-introduced strains were compared by HPLC. The results are shown in Table 2 (results of CJM2-type strains) and Table 3 (results of CJM3-type strains).
TABLE-US-00012 TABLE 1 Composition Concentration (per liter) Glucose 70 g Ammonium sulfate 25 g KH2PO4 1 g MgSO4•7H2O 0.5 g FeSO4•7H2O 5 mg MnSO4•8H2O 5 mg ZnSO4 5 mg Calcium carbonate 30 g Yeast Extract 2 g Methionine 0.3 g Threonine 1.5 g
TABLE-US-00013 TABLE 2 Sugar Produc- consump- tion tion Product amount Strains OD (g/L) (g/L) (g/L) CJM2 35.6 63.8 O-acetyl 12.3 pCL_Pcj1_metX homoserine CJM2 31.3 49.1 O-succinyl 2.7 pCL_Pcj1_metA(wt) homoserine CJM2 32.6 48.3 O-acetyl 2.5 pCL_Pcj1_metA EL homoserine CJM2 33.6 50.2 O-acetyl 2.0 pCL_Pcj1_metA ET homoserine CJM2 31.9 47.5 O-acetyl 3.1 pCL_Pcj1_metA EH homoserine CJM2 29.5 56.2 O-succinyl 11.3 pCL_Pcj1_metA(#11) homoserine CJM2 32.7 49.0 O-acetyl 7.8 pCL_Pcj1_metA(#11)EL homoserine CJM2 38 53.7 O-acetyl 6 pCL_Pcj1_metA(#11)ET homoserine CJM2 34.5 59.1 O-acetyl 11.1 pCL_Pcj1_metA(#11)EH homoserine
TABLE-US-00014 TABLE 3 Sugar Produc- consump- tion tion Product amount Strains OD (g/L) (g/L) (g/L) CJM3 17.2 67.0 O-acetyl 23.7 pCL_Pcj1_metX homoserine CJM3 18.8 60.5 O-succinyl 1.2 pCL_Pcj1_metA(wt) homoserine CJM3 18.5 60.5 O-acetyl 2.1 pCL_Pcj1_metA EL homoserine CJM3 18.0 61.0 O-acetyl 2.2 pCL_Pcj1_metA ET homoserine CJM3 17.8 62.2 O-acetyl 3.2 pCL_Pcj1_metA EH homoserine CJM3 14.6 67.0 O-succinyl 16.1 pCL_Pcj1_metA(#11) homoserine CJM3 17.1 63.2 O-acetyl 12.5 pCL_Pcj1_metA(#11)EL homoserine CJM3 18.2 65.1 O-acetyl 16.7 pCL_Pcj1_metA(#11)ET homoserine CJM3 19.0 67.8 O-acetyl 24.8 pCL_Pcj1_metA(#11)EH homoserine
[0128] As shown in Tables 2 and 3, only O-succinyl homoserine was produced by pCL_Pcj1_metA(wt) including the wild-type metA gene, but only O-acetyl homoserine was accumulated by the strains including three mutated metA genes of the present invention. That is, homoserine succinyltransferase activity of the polypeptide was modified to homoserine acetyltransferase activity by substitution of its amino acids.
[0129] Further, among the three mutants of CJM3-type strain, the strain (EL) prepared by substitution of glutamic acid for the amino acid at position 111 produced 2.1 g/L of O-acetyl homoserine, whereas the strain (EH) prepared by additional substitution of histidine for the amino acid at position 112 produced 3.2 g/L of O-acetyl homoserine, which is the highest yield of O-acetyl homoserine.
[0130] The strains expressing modified polypeptides having homoserine acetyltransferase activity resistant to feedback regulation by methionine also showed the same results. Specifically, the metA #11(EH) gene-introduced strain, which had a resistance to feedback regulation by methionine and substitutions of glutamic acid and histidine for the amino acids at position 111 and 112, produced the largest amount of O-acetyl homoserine (24.8 g/L), indicating that it accumulates O-acetyl homoserine at the similar level to that introduced with the foreign homoserine acetyltransferase gene (CJM3 pCL_Pcj1_metX, 23.7 g/L).
EFFECT OF THE INVENTION
[0131] According to the present invention, O-acetyl homoserine can be produced from homoserine without introduction of a foreign gene into a microorganism that expresses an enzyme which converts homoserine into O-succinyl homoserine, and the above O-acetyl homoserine can be used as a precursor for the production of methionine. Therefore, when the present invention is applied to the production of methionine for use in foods, it is advantageous in that the problems of anxiety and negative attitudes of consumers toward introduction of foreign genes and provision of proof of safety for the introduction of foreign genes can be solved.
Sequence CWU
1
1
44129DNAArtificial Sequenceforward primer 1aattgatatc atgccgattc gtgtgccgg
29234DNAArtificial Sequencereverse
primer 2aattaagctt ttaatccagc gttggattca tgtg
34327DNAArtificial Sequenceforward primer 3aattgatatc atgaccgccg
tgctcgc 27431DNAArtificial
Sequencereverse primer 4aattaagctt tcaactcctg agaaacgccc c
31535DNAArtificial Sequenceforward primer 5atgacaactt
ctcgtgcgcc tggtcaggaa attcg
35635DNAArtificial Sequencereverse primer 6cgaatttcct gaccaggcgc
acgagaagtt gtcat 35735DNAArtificial
Sequenceforward primer 7cgccgctggg cctggtgggg tttaatgatg tcgct
35835DNAArtificial Sequencereverse primer 8agcgacatca
ttaaacccca ccaggcccag cggcg
35935DNAArtificial Sequenceforward primer 9cacgtcacct cgacgctgag
tgtctgctgg gcggt 351035DNAArtificial
Sequencereverse primer 10accgcccagc agacactcag cgtcgaggtg acgtg
351144DNAArtificial Sequenceforward primer
11ttgtaactgg tgcgccgctg gaactggtgg ggtttaatga tgtc
441244DNAArtificial Sequencereverse primer 12gacatcatta aaccccacca
gttccagcgg cgcaccagtt acaa 441344DNAArtificial
Sequenceforward primer 13tgtaactggt gcgccgctgg aaaccgtggg gtttaatgat gtcg
441444DNAArtificial Sequencereverse primer
14cgacatcatt aaaccccacg gtttccagcg gcgcaccagt taca
441544DNAArtificial Sequenceforward primer 15tgtaactggt gcgccgctgg
aacatgtggg gtttaatgat gtcg 441644DNAArtificial
Sequencereverse primer 16cgacatcatt aaaccccaca tgttccagcg gcgcaccagt taca
4417309PRTArtificial SequenceMetA polypeptide having
homoserine O-succinyl transferase activity 17Met Pro Ile Arg Val Pro
Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1 5
10 15Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala
Ser Gly Gln Glu 20 25 30Ile
Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile 35
40 45Glu Thr Glu Asn Gln Phe Leu Arg Leu
Leu Ser Asn Ser Pro Leu Gln 50 55
60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr65
70 75 80Pro Ala Glu His Leu
Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln 85
90 95Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly
Ala Pro Leu Gly Leu 100 105
110Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu
115 120 125Glu Trp Ser Lys Asp His Val
Thr Ser Thr Leu Phe Val Cys Trp Ala 130 135
140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr
Arg145 150 155 160Thr Glu
Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr Arg Gly Phe
Asp Asp Ser Phe Leu Ala Pro His Ser 180 185
190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr
Asp Leu 195 200 205Glu Ile Leu Ala
Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro
Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp
245 250 255Pro Asp Val Pro Tyr
Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu
Phe Thr Asn Trp 275 280 285Leu Asn
Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu Asp30518309PRTArtificial
SequenceVariant polypeptide having homoserine O-acetyl transferase
activity, metA EL 18Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn
Phe Leu Arg1 5 10 15Glu
Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu 20
25 30Ile Arg Pro Leu Lys Val Leu Ile
Leu Asn Leu Met Pro Lys Lys Ile 35 40
45Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln
50 55 60Val Asp Ile Gln Leu Leu Arg Ile
Asp Ser Arg Glu Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu
Asp Ile Gln 85 90 95Asp
Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Glu Leu
100 105 110Val Glu Phe Asn Asp Val Ala
Tyr Trp Pro Gln Ile Lys Gln Val Leu 115 120
125Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp
Ala 130 135 140Val Gln Ala Ala Leu Asn
Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg145 150
155 160Thr Glu Lys Leu Ser Gly Val Tyr Glu His His
Ile Leu His Pro His 165 170
175Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser
180 185 190Arg Tyr Ala Asp Phe Pro
Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu 195 200
205Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe
Ala Ser 210 215 220Lys Asp Lys Arg Ile
Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala225 230
235 240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp
Val Glu Ala Gly Leu Asp 245 250
255Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr
260 265 270Pro Arg Ala Ser Trp
Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp 275
280 285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp
Leu Arg His Met 290 295 300Asn Pro Thr
Leu Asp30519309PRTArtificial SequenceVariant polypeptide having
homoserine O-acetyl transferase activity, metA ET 19Met Pro Ile Arg
Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1 5
10 15Glu Glu Asn Val Phe Val Met Thr Thr Ser
Arg Ala Ser Gly Gln Glu 20 25
30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile
35 40 45Glu Thr Glu Asn Gln Phe Leu Arg
Leu Leu Ser Asn Ser Pro Leu Gln 50 55
60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr65
70 75 80Pro Ala Glu His Leu
Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln 85
90 95Asp Gln Asn Phe Asp Gly Leu Ile Val Thr Gly
Ala Pro Leu Glu Thr 100 105
110Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu
115 120 125Glu Trp Ser Lys Asp His Val
Thr Ser Thr Leu Phe Val Cys Trp Ala 130 135
140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr
Arg145 150 155 160Thr Glu
Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr Arg Gly Phe
Asp Asp Ser Phe Leu Ala Pro His Ser 180 185
190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr
Asp Leu 195 200 205Glu Ile Leu Ala
Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro
Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp
245 250 255Pro Asp Val Pro Tyr
Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu
Phe Thr Asn Trp 275 280 285Leu Asn
Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu Asp30520309PRTArtificial
SequenceVariant polypeptide having homoserine O-acetyl transferase
activity, metA EH 20Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn
Phe Leu Arg1 5 10 15Glu
Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu 20
25 30Ile Arg Pro Leu Lys Val Leu Ile
Leu Asn Leu Met Pro Lys Lys Ile 35 40
45Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln
50 55 60Val Asp Ile Gln Leu Leu Arg Ile
Asp Ser Arg Glu Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu
Asp Ile Gln 85 90 95Asp
Gln Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Glu His
100 105 110Val Glu Phe Asn Asp Val Ala
Tyr Trp Pro Gln Ile Lys Gln Val Leu 115 120
125Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp
Ala 130 135 140Val Gln Ala Ala Leu Asn
Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg145 150
155 160Thr Glu Lys Leu Ser Gly Val Tyr Glu His His
Ile Leu His Pro His 165 170
175Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser
180 185 190Arg Tyr Ala Asp Phe Pro
Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu 195 200
205Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe
Ala Ser 210 215 220Lys Asp Lys Arg Ile
Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala225 230
235 240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp
Val Glu Ala Gly Leu Asp 245 250
255Pro Asp Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr
260 265 270Pro Arg Ala Ser Trp
Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp 275
280 285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp
Leu Arg His Met 290 295 300Asn Pro Thr
Leu Asp30521309PRTArtificial Sequencefeedback-resistance variant
polypeptide having hoserine O-acetyltransferase activity, met11A EL
21Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1
5 10 15Glu Glu Asn Val Phe Val
Met Thr Thr Ser Arg Ala Pro Gly Gln Glu 20 25
30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro
Lys Lys Ile 35 40 45Glu Thr Glu
Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln 50
55 60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu
Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln
85 90 95Asp Gln Asn Phe Asp Glu
Thr Ile Val Thr Gly Ala Pro Leu Glu Leu 100
105 110Val Gly Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile
Lys Gln Val Leu 115 120 125Glu Trp
Ser Lys Asp His Val Thr Ser Thr Leu Pro Val Cys Trp Ala 130
135 140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile
Pro Lys Gln Thr Arg145 150 155
160Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr
Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser 180
185 190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg
Asp Tyr Thr Asp Leu 195 200 205Glu
Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly
His Pro Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu
Asp 245 250 255Pro Asp Val
Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn
Leu Leu Phe Thr Asn Trp 275 280
285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu
Asp30522309PRTArtificial Sequencefeedback-resistance variant polypeptide
having hoserine O-acetyltransferase activity, met11A ET 22Met Pro
Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1 5
10 15Glu Glu Asn Val Phe Val Met Thr
Thr Ser Arg Ala Pro Gly Gln Glu 20 25
30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys
Ile 35 40 45Glu Thr Glu Asn Gln
Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln 50 55
60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg
Asn Thr65 70 75 80Pro
Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln
85 90 95Asp Gln Asn Phe Asp Glu Thr
Ile Val Thr Gly Ala Pro Leu Glu Thr 100 105
110Val Gly Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln
Val Leu 115 120 125 Glu Trp Ser
Lys Asp His Val Thr Ser Thr Leu Pro Val Cys Trp Ala 130
135 140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro
Lys Gln Thr Arg145 150 155
160Thr Glu Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr Arg
Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser 180
185 190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp
Tyr Thr Asp Leu 195 200 205Glu Ile
Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His
Pro Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp
245 250 255Pro Asp Val Pro
Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu
Leu Phe Thr Asn Trp 275 280 285Leu
Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu Asp30523309PRTArtificial
Sequencefeedback-resistance variant polypeptide having hoserine
O-acetyltransferase activity, met11A EH 23Met Pro Ile Arg Val Pro Asp Glu
Leu Pro Ala Val Asn Phe Leu Arg1 5 10
15Glu Glu Asn Val Phe Val Met Thr Thr Ser Arg Ala Pro Gly
Gln Glu 20 25 30Ile Arg Pro
Leu Lys Val Leu Ile Leu Asn Leu Met Pro Lys Lys Ile 35
40 45Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser
Asn Ser Pro Leu Gln 50 55 60Val Asp
Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu Ser Arg Asn Thr65
70 75 80Pro Ala Glu His Leu Asn Asn
Phe Tyr Cys Asn Phe Glu Asp Ile Gln 85 90
95Asp Gln Asn Phe Asp Glu Thr Ile Val Thr Gly Ala Pro
Leu Glu His 100 105 110Val Gly
Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile Lys Gln Val Leu 115
120 125Glu Trp Ser Lys Asp His Val Thr Ser Thr
Leu Pro Val Cys Trp Ala 130 135 140Val
Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile Pro Lys Gln Thr Arg145
150 155 160Thr Glu Lys Leu Ser Gly
Val Tyr Glu His His Ile Leu His Pro His 165
170 175Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu
Ala Pro His Ser 180 185 190Arg
Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu 195
200 205Glu Ile Leu Ala Glu Thr Glu Glu Gly
Asp Ala Tyr Leu Phe Ala Ser 210 215
220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala225
230 235 240Gln Thr Leu Ala
Gln Glu Phe Phe Arg Asp Val Glu Ala Gly Leu Asp 245
250 255Pro Asp Val Pro Tyr Asn Tyr Phe Pro His
Asn Asp Pro Gln Asn Thr 260 265
270Pro Arg Ala Ser Trp Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp
275 280 285Leu Asn Tyr Tyr Val Tyr Gln
Ile Thr Pro Tyr Asp Leu Arg His Met 290 295
300Asn Pro Thr Leu Asp30524930DNAArtificial Sequencepolynucleotide
coding variant polypeptide, metAEL 24atgccgattc gtgtgccgga
cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60tttgtgatga caacttctcg
tgcgtctggt caggaaattc gtccacttaa ggttctgatc 120cttaacctga tgccgaagaa
gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180tcacctttgc aggtcgatat
tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240cccgcagagc atctgaacaa
cttctactgt aactttgaag atattcagga tcagaacttt 300gacggtttga ttgtaactgg
tgcgccgctg gaactggtgg agtttaatga tgtcgcttac 360tggccgcaga tcaaacaggt
gctggagtgg tcgaaagatc acgtcacctc gacgctgttt 420gtctgctggg cggtacaggc
cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480accgaaaaac tctctggcgt
ttacgagcat catattctcc atcctcatgc gcttctgacg 540cgtggctttg atgattcatt
cctggcaccg cattcgcgct atgctgactt tccggcagcg 600ttgattcgtg attacaccga
tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660ctgtttgcca gtaaagataa
gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720caaacgctgg cgcaggaatt
tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780tataactatt tcccgcacaa
tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840ggtaatttac tgtttaccaa
ctggctcaac tattacgtct accagatcac gccatacgat 900ctacggcaca tgaatccaac
gctggattaa 93025930DNAArtificial
Sequencepolynucleotide coding variant polypeptide, metAET
25atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc
60tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccacttaa ggttctgatc
120cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac
180tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg
240cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt
300gacggtttga ttgtaactgg tgcgccgctg gaaaccgtgg agtttaatga tgtcgcttac
360tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgttt
420gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc
480accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg
540cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg
600ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat
660ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg
720caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg
780tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac
840ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcac gccatacgat
900ctacggcaca tgaatccaac gctggattaa
93026930DNAArtificial Sequencepolynucleotide coding variant polypeptide,
metAEH 26atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga
agaaaacgtc 60tttgtgatga caacttctcg tgcgtctggt caggaaattc gtccacttaa
ggttctgatc 120cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct
gctttcaaac 180tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc
gcgcaacacg 240cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga
tcagaacttt 300gacggtttga ttgtaactgg tgcgccgctg gaacatgtgg agtttaatga
tgtcgcttac 360tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc
gacgctgttt 420gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa
gcaaactcgc 480accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc
gcttctgacg 540cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt
tccggcagcg 600ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg
ggatgcatat 660ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga
atatgatgcg 720caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc
ggatgtaccg 780tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg
gcgtagtcac 840ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcac
gccatacgat 900ctacggcaca tgaatccaac gctggattaa
93027930DNAArtificial Sequencepolynucleotide coding variant
polypeptide, met11AEL 27atgccgattc gtgtgccgga cgagctaccc gccgtcaatt
tcttgcgtga agaaaacgtc 60tttgtgatga caacttctcg tgcgcctggt caggaaattc
gtccacttaa ggttctgatc 120cttaacctga tgccgaagaa gattgaaact gaaaatcagt
ttctgcgcct gctttcaaac 180tcacctttgc aggtcgatat tcagctgttg cgcatcgatt
cccgtgaatc gcgcaacacg 240cccgcagagc atctgaacaa cttctactgt aactttgaag
atattcagga tcagaacttt 300gacggtttga ttgtaactgg tgcgccgctg gaactggtgg
ggtttaatga tgtcgcttac 360tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc
acgtcacctc gacgctgtct 420gtctgctggg cggtacaggc cgcgctcaat atcctctacg
gcattcctaa gcaaactcgc 480accgaaaaac tctctggcgt ttacgagcat catattctcc
atcctcatgc gcttctgacg 540cgtggctttg atgattcatt cctggcaccg cattcgcgct
atgctgactt tccggcagcg 600ttgattcgtg attacaccga tctggaaatt ctggcagaga
cggaagaagg ggatgcatat 660ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg
gccatcccga atatgatgcg 720caaacgctgg cgcaggaatt tttccgcgat gtggaagccg
gactagaccc ggatgtaccg 780tataactatt tcccgcacaa tgatccgcaa aatacaccgc
gagcgagctg gcgtagtcac 840ggtaatttac tgtttaccaa ctggctcaac tattacgtct
accagatcac gccatacgat 900ctacggcaca tgaatccaac gctggattaa
93028930DNAArtificial Sequencepolynucleotide
coding variant polypeptide, met11AET 28atgccgattc gtgtgccgga
cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc 60tttgtgatga caacttctcg
tgcgcctggt caggaaattc gtccacttaa ggttctgatc 120cttaacctga tgccgaagaa
gattgaaact gaaaatcagt ttctgcgcct gctttcaaac 180tcacctttgc aggtcgatat
tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg 240cccgcagagc atctgaacaa
cttctactgt aactttgaag atattcagga tcagaacttt 300gacggtttga ttgtaactgg
tgcgccgctg gaaaccgtgg ggtttaatga tgtcgcttac 360tggccgcaga tcaaacaggt
gctggagtgg tcgaaagatc acgtcacctc gacgctgtct 420gtctgctggg cggtacaggc
cgcgctcaat atcctctacg gcattcctaa gcaaactcgc 480accgaaaaac tctctggcgt
ttacgagcat catattctcc atcctcatgc gcttctgacg 540cgtggctttg atgattcatt
cctggcaccg cattcgcgct atgctgactt tccggcagcg 600ttgattcgtg attacaccga
tctggaaatt ctggcagaga cggaagaagg ggatgcatat 660ctgtttgcca gtaaagataa
gcgcattgcc tttgtgacgg gccatcccga atatgatgcg 720caaacgctgg cgcaggaatt
tttccgcgat gtggaagccg gactagaccc ggatgtaccg 780tataactatt tcccgcacaa
tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac 840ggtaatttac tgtttaccaa
ctggctcaac tattacgtct accagatcac gccatacgat 900ctacggcaca tgaatccaac
gctggattaa 93029930DNAArtificial
Sequencepolynucleotide coding variant polypeptide, met11AEH
29atgccgattc gtgtgccgga cgagctaccc gccgtcaatt tcttgcgtga agaaaacgtc
60tttgtgatga caacttctcg tgcgcctggt caggaaattc gtccacttaa ggttctgatc
120cttaacctga tgccgaagaa gattgaaact gaaaatcagt ttctgcgcct gctttcaaac
180tcacctttgc aggtcgatat tcagctgttg cgcatcgatt cccgtgaatc gcgcaacacg
240cccgcagagc atctgaacaa cttctactgt aactttgaag atattcagga tcagaacttt
300gacggtttga ttgtaactgg tgcgccgctg gaacatgtgg ggtttaatga tgtcgcttac
360tggccgcaga tcaaacaggt gctggagtgg tcgaaagatc acgtcacctc gacgctgtct
420gtctgctggg cggtacaggc cgcgctcaat atcctctacg gcattcctaa gcaaactcgc
480accgaaaaac tctctggcgt ttacgagcat catattctcc atcctcatgc gcttctgacg
540cgtggctttg atgattcatt cctggcaccg cattcgcgct atgctgactt tccggcagcg
600ttgattcgtg attacaccga tctggaaatt ctggcagaga cggaagaagg ggatgcatat
660ctgtttgcca gtaaagataa gcgcattgcc tttgtgacgg gccatcccga atatgatgcg
720caaacgctgg cgcaggaatt tttccgcgat gtggaagccg gactagaccc ggatgtaccg
780tataactatt tcccgcacaa tgatccgcaa aatacaccgc gagcgagctg gcgtagtcac
840ggtaatttac tgtttaccaa ctggctcaac tattacgtct accagatcac gccatacgat
900ctacggcaca tgaatccaac gctggattaa
93030134DNAArtificial Sequencepro promoter nucletotide 30tcgagcatag
catttttatc cataagatta gcggatctaa cctttacaat tgtgagcgct 60cacaattatg
atagattcaa ttgtgagcgg ataacaattt cacacagaat tcattaaaga 120ggagaaaggt
acat
1343161DNAArtificial Sequenceprimer 31aggggcttca tccgaattgc gccattgttg
caatggcggt gctggagctg cttcgaagtt 60c
613242DNAArtificial Sequenceprimer
32gatattcata tggaccatgg ctcgagcata gcatttttat cc
423342DNAArtificial Sequenceprimer 33ggataaaaat gctatgctcg agccatggtc
catatgaata tc 423460DNAArtificial Sequenceprimer
34cgatgttggc aggaatggtg tgtttgtgaa tttggctcat atgtaccttt ctcctcttta
603520DNAArtificial Sequenceforward primer 35atgagtataa aagagcaaac
203619DNAArtificial
Sequencereverse primer 36ttatttgcgt agtctgacc
193737DNAArtificial Sequenceforward primer
37ggaaaagtac aaccgccgcc gtattgcagg cgctatt
373837DNAArtificial Sequencereverse primer 38aatagcgcct gcaatacggc
ggcggttgta cttttcc 3739930DNAArtificial
SequencemetA nucleotide sequence 39atgccgattc gtgtgccgga cgagctaccc
gccgtcaatt tcttgcgtga agaaaacgtc 60tttgtgatga caacttctcg tgcgtctggt
caggaaattc gtccacttaa ggttctgatc 120cttaacctga tgccgaagaa gattgaaact
gaaaatcagt ttctgcgcct gctttcaaac 180tcacctttgc aggtcgatat tcagctgttg
cgcatcgatt cccgtgaatc gcgcaacacg 240cccgcagagc atctgaacaa cttctactgt
aactttgaag atattcagga tcagaacttt 300gacggtttga ttgtaactgg tgcgccgctg
ggcctggtgg agtttaatga tgtcgcttac 360tggccgcaga tcaaacaggt gctggagtgg
tcgaaagatc acgtcacctc gacgctgttt 420gtctgctggg cggtacaggc cgcgctcaat
atcctctacg gcattcctaa gcaaactcgc 480accgaaaaac tctctggcgt ttacgagcat
catattctcc atcctcatgc gcttctgacg 540cgtggctttg atgattcatt cctggcaccg
cattcgcgct atgctgactt tccggcagcg 600ttgattcgtg attacaccga tctggaaatt
ctggcagaga cggaagaagg ggatgcatat 660ctgtttgcca gtaaagataa gcgcattgcc
tttgtgacgg gccatcccga atatgatgcg 720caaacgctgg cgcaggaatt tttccgcgat
gtggaagccg gactagaccc ggatgtaccg 780tataactatt tcccgcacaa tgatccgcaa
aatacaccgc gagcgagctg gcgtagtcac 840ggtaatttac tgtttaccaa ctggctcaac
tattacgtct accagatcac gccatacgat 900ctacggcaca tgaatccaac gctggattaa
93040316PRTArtificial Sequencefeedback
resistant coaA, coaA R106A 40Met Ser Ile Lys Glu Gln Thr Leu Met Thr Pro
Tyr Leu Gln Phe Asp1 5 10
15Arg Asn Gln Trp Ala Ala Leu Arg Asp Ser Val Pro Met Thr Leu Ser
20 25 30Glu Asp Glu Ile Ala Arg Leu
Lys Gly Ile Asn Glu Asp Leu Ser Leu 35 40
45Glu Glu Val Ala Glu Ile Tyr Leu Pro Leu Ser Arg Leu Leu Asn
Phe 50 55 60Tyr Ile Ser Ser Asn Leu
Arg Arg Gln Ala Val Leu Glu Gln Phe Leu65 70
75 80Gly Thr Asn Gly Gln Arg Ile Pro Tyr Ile Ile
Ser Ile Ala Gly Ser 85 90
95Val Ala Val Gly Lys Ser Thr Thr Ala Arg Val Leu Gln Ala Leu Leu
100 105 110Ser Arg Trp Pro Glu His
Arg Arg Val Glu Leu Ile Thr Thr Asp Gly 115 120
125Phe Leu His Pro Asn Gln Val Leu Lys Glu Arg Gly Leu Met
Lys Lys 130 135 140Lys Gly Phe Pro Glu
Ser Tyr Asp Met His Arg Leu Val Lys Phe Val145 150
155 160Ser Asp Leu Lys Ser Gly Val Pro Asn Val
Thr Ala Pro Val Tyr Ser 165 170
175His Leu Ile Tyr Asp Val Ile Pro Asp Gly Asp Lys Thr Val Val Gln
180 185 190Pro Asp Ile Leu Ile
Leu Glu Gly Leu Asn Val Leu Gln Ser Gly Met 195
200 205Asp Tyr Pro His Asp Pro His His Val Phe Val Ser
Asp Phe Val Asp 210 215 220Phe Ser Ile
Tyr Val Asp Ala Pro Glu Asp Leu Leu Gln Thr Trp Tyr225
230 235 240Ile Asn Arg Phe Leu Lys Phe
Arg Glu Gly Ala Phe Thr Asp Pro Asp 245
250 255Ser Tyr Phe His Asn Tyr Ala Lys Leu Thr Lys Glu
Glu Ala Ile Lys 260 265 270Thr
Ala Met Thr Leu Trp Lys Glu Ile Asn Trp Leu Asn Leu Lys Gln 275
280 285Asn Ile Leu Pro Thr Arg Glu Arg Ala
Ser Leu Ile Leu Thr Lys Ser 290 295
300Ala Asn His Ala Val Glu Glu Val Arg Leu Arg Lys305 310
31541309PRTArtificial SequenceECOHS, E. coli O9H4(strain
HS) 41Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1
5 10 15Glu Glu Asn Val Phe
Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu 20
25 30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met
Pro Lys Lys Ile 35 40 45Glu Thr
Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln 50
55 60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg
Glu Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln
85 90 95Glu Gln Asn Phe Asp
Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu 100
105 110Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile
Lys Gln Val Leu 115 120 125Glu Trp
Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala 130
135 140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile
Pro Lys Gln Thr Arg145 150 155
160Thr Asp Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr
Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser 180
185 190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg
Asp Tyr Thr Asp Leu 195 200 205Glu
Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly
His Pro Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Tyr Phe Arg Asp Val Glu Ala Gly Leu
Gly 245 250 255Pro Glu Val
Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn
Leu Leu Phe Thr Asn Trp 275 280
285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu
Asp30542309PRTArtificial SequenceECO24, E. coli O139 H28(strain E24377A)
42Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu Arg1
5 10 15Glu Glu Asn Val Phe Val
Met Thr Thr Ser Arg Ala Ser Gly Gln Glu 20 25
30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn Leu Met Pro
Lys Lys Ile 35 40 45Glu Thr Glu
Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln 50
55 60Val Asp Ile Gln Leu Leu Arg Ile Asp Ser Arg Glu
Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp Ile Gln
85 90 95Glu Gln Asn Phe Asp Gly
Leu Ile Val Thr Gly Ala Pro Leu Gly Leu 100
105 110Val Glu Phe Asn Asp Val Ala Tyr Trp Pro Gln Ile
Lys Gln Val Leu 115 120 125Glu Trp
Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala 130
135 140Val Gln Ala Ala Leu Asn Ile Leu Tyr Gly Ile
Pro Lys Gln Thr Arg145 150 155
160Thr Asp Lys Leu Ser Gly Val Tyr Glu His His Ile Leu His Pro His
165 170 175Ala Leu Leu Thr
Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser 180
185 190Arg Tyr Ala Asp Phe Pro Ala Ala Leu Ile Arg
Asp Tyr Thr Asp Leu 195 200 205Glu
Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe Ala Ser 210
215 220Lys Asp Lys Arg Ile Ala Phe Val Thr Gly
His Pro Glu Tyr Asp Ala225 230 235
240Gln Thr Leu Ala Gln Glu Tyr Phe Arg Asp Val Glu Ala Gly Leu
Gly 245 250 255Pro Glu Val
Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Thr 260
265 270Pro Arg Ala Ser Trp Arg Ser His Gly Asn
Leu Leu Phe Thr Asn Trp 275 280
285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp Leu Arg His Met 290
295 300Asn Pro Thr Leu
Asp30543309PRTArtificial SequenceECO57, E. coli O157H7, E. coli(strain
ATCC8739) 43Met Pro Ile Arg Val Pro Asp Glu Leu Pro Ala Val Asn Phe Leu
Arg1 5 10 15Glu Glu Asn
Val Phe Val Met Thr Thr Ser Arg Ala Ser Gly Gln Glu 20
25 30Ile Arg Pro Leu Lys Val Leu Ile Leu Asn
Leu Met Pro Lys Lys Ile 35 40
45Glu Thr Glu Asn Gln Phe Leu Arg Leu Leu Ser Asn Ser Pro Leu Gln 50
55 60Val Asp Ile Gln Leu Leu Arg Ile Asp
Ser Arg Glu Ser Arg Asn Thr65 70 75
80Pro Ala Glu His Leu Asn Asn Phe Tyr Cys Asn Phe Glu Asp
Ile Gln 85 90 95Glu Gln
Asn Phe Asp Gly Leu Ile Val Thr Gly Ala Pro Leu Gly Leu 100
105 110Val Glu Phe Asn Asp Val Ala Tyr Trp
Pro Gln Ile Lys Gln Val Leu 115 120
125Glu Trp Ser Lys Asp His Val Thr Ser Thr Leu Phe Val Cys Trp Ala
130 135 140Val Gln Ala Ala Leu Asn Ile
Leu Tyr Gly Ile Pro Lys Gln Thr Arg145 150
155 160Thr Asp Lys Leu Ser Gly Val Tyr Glu His His Ile
Leu His Pro His 165 170
175Ala Leu Leu Thr Arg Gly Phe Asp Asp Ser Phe Leu Ala Pro His Ser
180 185 190Arg Tyr Ala Asp Phe Pro
Ala Ala Leu Ile Arg Asp Tyr Thr Asp Leu 195 200
205Glu Ile Leu Ala Glu Thr Glu Glu Gly Asp Ala Tyr Leu Phe
Ala Ser 210 215 220Lys Asp Lys Arg Ile
Ala Phe Val Thr Gly His Pro Glu Tyr Asp Ala225 230
235 240Gln Thr Leu Ala Gln Glu Tyr Phe Arg Asp
Val Glu Ala Gly Leu Asp 245 250
255Pro Glu Val Pro Tyr Asn Tyr Phe Pro His Asn Asp Pro Gln Asn Lys
260 265 270Pro Arg Ala Ser Trp
Arg Ser His Gly Asn Leu Leu Phe Thr Asn Trp 275
280 285Leu Asn Tyr Tyr Val Tyr Gln Ile Thr Pro Tyr Asp
Leu Arg His Met 290 295 300Asn Pro Thr
Leu Asp305442464DNAArtificial SequencemetX Deinococcus radiodurans
44atgcgagtgt tgaagttcgg cggtacatca gtggcaaatg cagaacgttt tctgcgtgtt
60gccgatattc tggaaagcaa tgccaggcag gggcaggtgg ccaccgtcct ctctgccccc
120gccaaaatca ccaaccacct ggtggcgatg attgaaaaaa ccattagcgg ccaggatgct
180ttacccaata tcagcgatgc cgaacgtatt tttgccgaac ttttgacggg actcgccgcc
240gcccagccgg ggttcccgct ggcgcaattg aaaactttcg tcgatcagga atttgcccaa
300ataaaacatg tcctgcatgg cattagtttg ttggggcagt gcccggatag catcaacgct
360gcgctgattt gccgtggcga gaaaatgtcg atcgccatta tggccggcgt attagaagcg
420cgcggtcaca acgttactgt tatcgatccg gtcgaaaaac tgctggcagt ggggcattac
480ctcgaatcta ccgtcgatat tgctgagtcc acccgccgta ttgcggcaag ccgcattccg
540gctgatcaca tggtgctgat ggcaggtttc accgccggta atgaaaaagg cgaactggtg
600gtgcttggac gcaacggttc cgactactct gctgcggtgc tggctgcctg tttacgcgcc
660gattgttgcg agatttggac ggacgttgac ggggtctata cctgcgaccc gcgtcaggtg
720cccgatgcga ggttgttgaa gtcgatgtcc taccaggaag cgatggagct ttcctacttc
780ggcgctaaag ttcttcaccc ccgcaccatt acccccatcg cccagttcca gatcccttgc
840ctgattaaaa ataccggaaa tcctcaagca ccaggtacgc tcattggtgc cagccgtgat
900gaagacgaat taccggtcaa gggcatttcc aatctgaata acatggcaat gttcagcgtt
960tctggtccgg ggatgaaagg gatggtcggc atggcggcgc gcgtctttgc agcgatgtca
1020cgcgcccgta ttttcgtggt gctgattacg caatcatctt ccgaatacag catcagtttc
1080tgcgttccac aaagcgactg tgtgcgagct gaacgggcaa tgcaggaaga gttctacctg
1140gaactgaaag aaggcttact ggagccgctg gcagtgacgg aacggctggc cattatctcg
1200gtggtaggtg atggtatgcg caccttgcgt gggatctcgg cgaaattctt tgccgcactg
1260gcccgcgcca atatcaacat tgtcgccatt gctcagggat cttctgaacg ctcaatctct
1320gtcgtggtaa ataacgatga tgcgaccact ggcgtgcgcg ttactcatca gatgctgttc
1380aataccgatc aggttatcga agtgtttgtg attggcgtcg gtggcgttgg cggtgcgctg
1440ctggagcaac tgaagcgtca gcaaagctgg ctgaagaata aacatatcga cttacgtgtc
1500tgcggtgttg ccaactcgaa ggctctgctc accaatgtac atggccttaa tctggaaaac
1560tggcaggaag aactggcgca agccaaagag ccgtttaatc tcgggcgctt aattcgcctc
1620gtgaaagaat atcatctgct gaacccggtc attgttgact gcacttccag ccaggcagtg
1680gcggatcaat atgccgactt cctgcgcgaa ggtttccacg ttgtcacgcc gaacaaaaag
1740gccaacacct cgtcgatgga ttactaccat cagttgcgtt atgcggcgga aaaatcgcgg
1800cgtaaattcc tctatgacac caacgttggg gctggattac cggttattga gaacctgcaa
1860aatctgctca atgcaggtga tgaattgatg aagttctccg gcattctttc tggttcgctt
1920tcttatatct tcggcaagtt agacgaaggc atgagtttct ccgaggcgac cacgctggcg
1980cgggaaatgg gttataccga accggacccg cgagatgatc tttctggtat ggatgtggcg
2040cgtaaactat tgattctcgc tcgtgaaacg ggacgtgaac tggagctggc ggatattgaa
2100attgaacctg tgctgcccgc agagtttaac gccgagggtg atgttgccgc ttttatggcg
2160aatctgtcac aactcgacga tctctttgcc gcgcgcgtgg cgaaggcccg tgatgaagga
2220aaagttttgc gctatgttgg caatattgat gaagatggcg tctgccgcgt gaagattgcc
2280gaagtggatg gtaatgatcc gctgttcaaa gtgaaaaatg gcgaaaacgc cctggccttc
2340tatagccact attatcagcc gctgccgttg gtactgcgcg gatatggtgc gggcaatgac
2400gttacagctg ccggtgtctt tgctgatctg ctacgtaccc tctcatggaa gttaggagtc
2460tgaa
2464
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